The present invention relates to novel substituted phenyl derivatives which are potent chloride channel blockers and as such useful in the treatment of sickle cell anaemia, brain oedema following ischaemia or tumours, diarrhea, hypertension (diuretic), bone metabolic disorders, osteoclast associated disorders and for the reduction of the intraocular pressure for the treatment of disorders such as glaucoma.
Chloride channels serve a wide variety of specific cellular functions. Thus, chloride channels contribute to the normal function of skeletal and smooth muscle cells. Blockers of chloride channels are known to be useful in the treatment of brain oedema following ischaemia or tumours, diarrhea, hypertension (diuretic), sickle cell anaemia, bone metabolic disorders, bone metastasizing cancers and in general osteoclast associated disorder as well as for the reduction of the intraocular pressure in disorders such as glaucoma. The compounds of the invention may also be useful in the treatment of allergic and inflammatory conditions and for the promotion of wound healing.
Sickle Cell Anaemia
The use of blockers of chloride channels for the treatment sickle-cell anaemia form a new therapeutic approach.
Sickle cell anaemia and the existence of sickle haemoglobin was the first genetic disease to be understood at the molecular level. The genetic defect underlying sickle cell anaemia causes the substitution of a single amino acid resulting in a mutant haemoglobin, sickle haemoglobin.
The physical manifestations of sickle cell disease is anaemia and painful ischaemic crises due to occlusion of the microcirculation by deformed erythrocytes (sickle cells). The primary cause of sickle erythrocyte deformation and distortion (or sickling) is a reversible polymerisation and gelation of sickle haemoglobin induced at the low oxygen tensions prevalent in metabolically active tissues. Sickle cells are also characterised by an enhanced cation permeability, resulting in cation depletion and cellular dehydration. Since the delay time for the polymerisation has been described as an extremely steep function of the sickle haemoglobin concentration itself, any decrease in cell volume will greatly increase the probability of sickling and thereby of vessel occlusion. Compounds which blocks the deoxygenation induced salt and volume (water) loss may delay the sickling process enough to avoid occlusion upon the passage of the sickle erythrocyte through metabolically active tissue. It has been estimated that a delay time of only 10 sec may suffice.
Several membrane ion channels and transporters present in normal erythrocytes has been suggested to participate in the altered membrane permeabilities of sickle cells. The favoured hypothesis has been stimulation of the Ca2+-activated K+-channel and several blockers of this channel has been suggested as therapeutic agents for the treatment of sickle-cell anaemia (Effects of Cetiedil on Monovalent Cation Permeability in the Erythrocyte: An explanation for the Efficacy of Cetiedil in the treatment of Sickle Cell Anaemia, Berkowitz, L. R., Orringer, E. P., Blood cells, (283-288 (1982) and U.S. Pat. No. 5,273,992).
Since, K+ efflux through a K-channel must be followed by an equal efflux of Clxe2x88x92 to maintain electroneutrality, blockade of erythrocyte chloride channels should be as effective as blocking the K-channels itself. An advantage to the use of chloride channel blockers is that salt loss which may occur due to activation of unknown K-channel types will indirectly be blocked too.
The compounds of the present invention are potent blockers of chloride channels as measured by concomitant measurements of conductive netfluxes of chloride and membrane potentials in suspensions of erythrocytes, and the compounds are therefore predicted to be useful in the treatment of sickle-cell disease.
Osteoporosis and Other Osteoclast Associated Disorders
The bone tissue is constantly renewed by the controlled activity of two cell types, osteoblasts, which lay down the new bone mass, and osteoclasts, which degrade and reabsorb the bone tissue by secretion of proteolytic enzymes such as cathepsin as well as acid, in particular HCl onto the bone surface. In osteoporosis the balance between the degradation and the synthesis is severely disturbed, which results in a progressive loss of bone material and gradual weakening of the skeleton. Clinically, hormone replacement studies has shown that the decline in estrogen levels at the onset of menopause is an important hormonal factor for the triggering of the disease. In vitro studies has shown that the osteoclasts are important targets cells for estrogen (i.e. Mano et al., Biochem. Biophys. Res. Commun. 223(3), 637-642, 1996) and that the hormone inhibits the bone reabsorbing activity of osteoclasts via induction of osteoclast apoptosis (Kameda et al., J. Exp. Med., 186(4), 489-495, 1997) and/or via altered resorbtion capacity of the individual cells. Thus, the major estrogene effect on bone metabolism seems to be an inhibition of bone degradation by a direct effect on the osteoclasts.
Osteoclast Physiology
As an alternative to hormone replacement down regulation of osteoclast acid producing activity by modulators of membrane transporters is an attractive, but hitherto clinical untested possibility. The physiological process whereby the osteoclast secrete HClxe2x80x94a key event in bone reabsorbtionxe2x80x94is relatively well understood and is conceptually similar to epithelial transport. Like epithelia cells osteoclasts are morphologically highly polarised cells with membrane transporters asymmetrically distributed between the bone-facing ruffled membrane and the smooth outer membrane. At the border between the ruffled and smooth membrane segments the osteoclast is tightly attached to the bone surface, thus creating a sealed cavity between the cell and the bone surface. Pits are formed beneath the cavity as a result of HCl-induced demineralisation and enzymatic break-down of the bone matrix.
The ultimate event in the osteoclast HCl secretion across the ruffled membrane is an active transport of H+ by a vacuolar-type proton pump and a passive transport of Clxe2x88x92 mediated via an outwardly rectifying Cl-channel. Due to HCl secretion the intracellular pH tend to increase and Clxe2x88x921 tend to decrease, whichxe2x80x94if allowed to occurxe2x80x94would quickly lead to cessation of acid secretion. Osteoclasts posses two important back-up systems aimed at maintaining a constant supply of intracellular H+ and Clxe2x88x92 for the ruffled membrane transporters. First, the cell contains very high concentrations of the cytosolic enzyme carbonic anhydrase II, which catalyses the slow normally quite slow hydration of CO2 to H2CO3, a molecule which spontaneously dissociate to form H+ and HCOxe2x88x923. Second, the osteoclast outer membrane is packed with transporters (AE2), which mediate obligatory Clxe2x88x92/HCOxe2x88x923 exchange. Hence, HCOxe2x88x923 produced by the carbonic anhydrase exzyme is exchanged with extracellular Clxe2x88x92. Apart from erythrocytes, osteoclasts are the mammalian cell type with the highest expression level of this protein.
In conclusion, the proton pump and the Cl-channel are fed by H+ and Clxe2x88x92, respectively, via the concerted activity of the carbonic anhydrase and the anion exchanger.
Possible Pharmacological Intervention
In principle, any of the four proteins described above which are directly involved in the transcellular secretion of HCl are valid targets for interference with the resorptive properties of osteoclasts.
Direct block of the proton pump is achievable with the antibiotic bafilomycin A1, which is an extremely potent, reversible inhibitor, whereas omeprazolxe2x80x94an irreversible inhibitor of the proton pump responsible for acid production in the stomachxe2x80x94is ineffective. In vitro bafilomycin A1 completely eliminates bone resorbtion in the bone slice assay pit formation test (Ohba et al, FEBS lett. 387(2-3), 175-178, 1996). In vivo the compound depresses bone degration in growing yound rats (Keeling et al, Ann. N. Y. Acad. Sci., 834, 600-608 1997). The general applicability of the compound is limited due to its toxicity, which may be due to undesired inhibition of proton pumps in other areas of the body. Subtypes of the vacuolar proton pump exists, however it is not known if it will be possible to obtain pharmacological selectivity between these isoforms.
Inhibition of the carbonic anhydrase enzyme with acetacolamide is effective in vitro in the pit formation assay (Ohba et al, FEBS lett. 387(2-3), 175-178, 1996). Various inhibitors of the kidney carbonic anhydrase enzymes has previously been used as diuretic agents.
It is well established that osteoclast ability for bone resorbtion is highly correleated with the expression of the ruffled membrane Cl-channel (Schlesinger et. al. Jour. Biol. Chem., 272(30), 18636-18643, 1997). Block of the ruffled border Cl-channel is only achievable with very high concentratios of stilbene-sulfonates like DIDS, which makes firm conclusions about the efficacy of a selective block questionable, especially since the stilbenes most likely will block the anion exchanger even better than the Cl-channel.
A blockade of the anion exchanger in osteoclasts will also be a regulatory site for osteoclast activity optionally in combination with blockade of the chloride channel as a concomitant blockade of both channels or a blockade of either channel alone.
It is also well established that osteoclasts are involved in other bone-tissue related disorders (i.e. Clohisy et. al., J. Orthop. Res., 14(3), 396-702, 1996) and intervention with the osteoclast activity is very likely to prevent these types of disorders. The compounds of the present invention administered alone as well as in combination with other well known treatments of bone metabolism related diseases will have an effect on these diseases.
The compounds of the present invention are potent blockers of chloride channels and also the anion exchanger, as measured by concomitant measurements of conductive netfluxes of chloride and membrane potentials in suspensions of erythrocytes, and the compounds are therefore predicted to be useful in the treatment of sickle-cell disease, osteoporosis as well as other osteoclast associated disorders.
Several chloride channel blockers and the use thereof have already been described:
Pflxc3xcgers Arch (1986), 407 (suppl. 2), pages 128-141 describes several compounds with chloride channel blocking activity. A very potent compound described herein is 5-nitro-2-(3-phenylpropylamino)benzoic acid. The use of chloride channel blockers for the treatment of sickle cell anaemia is not disclosed herein.
U.S. Pat. No. 4,889,612 describes Calixarene derivatives and their use as chloride channel blockers.
U.S. Pat. No. 4,994,493 describes certain 5-nitrobenzoic acid derivatives and their use in the treatment of cerebral oedema.
WO 96/16647 describes the use of chloride channel blockers for reduction of the intraocular pressure and specifically the use of chloride channel blockers for the treatment of glaucoma.
WO 97/45400 describes other phenyl derivatives as chloride channels blockers;
The present invention relates to a series of substituted phenyl derivatives which are potent chloride channel blockers, and their use in the treatment of sickle-cell anaemia and osteoporosis as well as other bone metabolic disorders. The compounds of the present invention are novel and differs as well in the substitution patterns as in pharmacodynamic properties such as kinetic behaviour, bioavailability, solubility and efficacy.
It is an object of the present invention to provide novel substituted phenyl derivatives and pharmaceutically acceptable salts thereof which are useful in the treatment of disorders or diseases responsive to the blockade of chloride channels.
Still another object of the present invention is to provide a method of treating disorders or diseases responsive to the blockade of chloride channels, such as for example brain oedema following ischaemia or tumours, diarrhea, hypertension (diuretic), glaucoma and in particular sickle cell anaemia and osteoporosis.
The invention then comprises, inter alia, alone or in combination:
A compound having the formula 
or a pharmaceutically acceptable salt thereof
wherein one of R1, R2 and R3 is a acidic functional group having a pKa value below 8 or a group which is convertible in vivo to such a group;
R4, R5 and the other two substituents R1, R2 and R3 are each independently selected from hydrogen; alkyl; alkoxy; hydroxy; halogen; trifluoromethyl; cyano; nitro; amino; alkylamino, NHCOR9, CO2R9, xe2x80x94CON(R9)2, xe2x80x94NHSO2xe2x80x94R9, xe2x80x94SO2N(R9)2 wherein R9 represents hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl or R9 comprises a 5-8 membered ring optionally containing double bonds and optionally containing one or two heteroatoms, which can be substituted by alkyl or acyl; or (R9)2 together with the heteroatom to which it is connected represents a 5-8 membered ring optionally containing double bonds and optionally containing another heteroatom which can be substituted by alkyl or acyl; aryl, phenylamino, phenoxy or heteroaryl wherein the phenyl, aryl or heteroaryl group may be substituted one or more times with substituents selected from alkyl, hydroxy, alkoxy, halogen, trifluoromethyl, cyano, nitro, amino, NHCOR9, CO2R9, xe2x80x94CON(R9)2, xe2x80x94NHSO2xe2x80x94R9, xe2x80x94SO2N(R9)2 wherein R9 is as defined above; or R3 and R4 or R4 and R5 together form a 4-8 membered saturated or unsaturated ring and the other substituents R1, R2, R3, R4 and R5 is as defined above;
Y represents 
xe2x80x83wherein R0 and R00 independently represents hydrogen or lower alkyl;
X represents O or S;
R6 is hydrogen, or alkyl;
R11, R12, R13, R14 and R15 are each independently selected from hydrogen; alkyl; alkoxy; hydroxy; halogen; trifluoromethyl; cyano; nitro; amino; alkylamino; xe2x80x94COOR7;
xe2x80x94NHSO2-alkyl; xe2x80x94SO2N(R7)2; xe2x80x94SO2OR7; xe2x80x94COxe2x80x94R7; aryl, phenylamino, phenoxy or heteroaryl wherein the phenyl, aryl or heteroaryl group may be substituted one or more times with substituents selected from alkyl, hydroxy, alkoxy, halogen, trifluoromethyl, cyano, nitro, amino and alkylamino, xe2x80x94COOR7; CON(R7)2. xe2x80x94NHSO2xe2x80x94R7; xe2x80x94SO2N(R7)2; xe2x80x94SO2OR7; xe2x80x94COxe2x80x94R7, or one of R11 and R12, R12 and R13, R13 and R14 and R14 and R15 together form a 4-8 membered saturated or unsaturated ring and the other substituents R11, R12, R13, R14 and R15 is as defined above and wherein R7 represents hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl or R7 comprises a 5-8 membered ring optionally containing double bonds and optionally containing one or two heteroatoms which can be substituted by alkyl or acyl; or (R7)2 together with the heteroatom to which it is connected represents a 5-8 membered ring optionally containing double bonds and optionally containing another heteroatom which can be substituted by alkyl or acyl;
a method for the preparation of a compound as above, comprising:
a) reacting a compound having the formula 
xe2x80x83wherein W is O, or S and R11, R12, R13, R14 and R15 is as defined above, with a compound having the formula 
xe2x80x83wherein R1, R2, R3, R4, R5 and R6 is as defined above, or
b) reacting a compound having the formula 
wherein X, Y, R6, R11, R12, R13, R14 and R15 is as defined above, with a compound having the formula 
wherein Hal is halogen and R1, R2, R3, R4 and R5 is as defined above,
whereafter optionally the compound obtained is converted to another compound of the invention and/or a pharmaceutically acceptable salt thereof is formed using conventional methods;
a chloride channel blocker as above optionally combined with known therapeutic treatment of bone metabolic disorders;
a pharmaceutical composition comprising a therapeutically effective amount of a compound as any above or a pharmaceutically acceptable salt thereof together with at least one pharmaceutically acceptable carrier or diluent;
the use of a compound as above for the preparation of a medicament for the treatment of a disorder or disease of a living animal body, including a human, which disorder or disease is responsive to the blockade of chloride channels;
a method for the treatment of a disorder or disease of a living animal body, including a human, which disorder or disease is responsive to the blockade of chloride channels, comprising administering to such a living animal body in need thereof a therapeutically effective amount of a compound as above;
The invention comprises in a preferred aspect a compound of the formula 
wherein one of R1, R2 and R3 is
3-hydroxy-4-oxo-pyranyl, 2-hydroxy-4-oxo-pyrimidyl, 3,5-dioxo-1,2,4-oxadiazolidinyl, 2,4-dioxo-imidazolidinyl, 2,5-dioxo-3-hydroxy-pyrrolyl, 2,5-dioxo-pyrrolidinyl, 2,4-dioxo-1,3-thiazolidinyl, 3-hydroxy-isoxazolyl, 5-hydroxy-isoxazolyl, 3-hydroxy-isothiazolyl, 3-hydroxy-1,2,5-thiadiazolyl, tetrazolyl, 3-hydroxy-triazolyl, 3-hydroxy-pyrazolyl, 2-hydroxy-1,3,4-oxadiazolyl or 2-hydroxy-3,4-dioxo-cyclobutenyl, NH2, xe2x80x94COOR16, xe2x80x94CH2COOR16, xe2x80x94CON(R16)2, xe2x80x94NHSO2xe2x80x94R16, xe2x80x94SO2N(R16)2, xe2x80x94SO2R16, xe2x80x94PO3RH, xe2x80x94PO2NH2, xe2x80x94CONHOH, xe2x80x94CONHCN, xe2x80x94CONHSO2R16 and xe2x80x94CONHNH2, wherein R16 is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl or R16 comprises a 5-8 membered ring optionally containing double bonds and optionally containing one or two heteroatoms, or (R16)2 together with the heteroatom to which it is connected represents a 5-8 membered ring optionally containing double bonds and optionally containing another heteroatom; and the other of R1, R2 and R3 is as defined above;
A preferred aspect of the invention comprises compound as above, wherein one of R1, R2 and R3 represents COOH, CH2COOR16, CON(R16)2, tetrazolyl, methyltetrazolyl, NHSO2R16, CO2R16, CO2N(R16)2, SO2N(R16)2, CONHSO2R16; SO2OR16, wherein R16 is as defined above;
Another preferred aspect of the invention comprises a compound as above wherein R4, R5 and the remaining of R1, R2 or R3 independently represents hydrogen, alkyl, nitro, amino, alkylamino, CO2R9, CF3, alkyl, halogen, hydroxy, alkoxy, NHCOR9, xe2x80x94CON(R9)2, aryl optionally substituted with CON(R9)2, NHCOR9, SO2N(R9)2, CO2R9, wherein R9 is as defined above;
Yet another preferred aspect of the invention comprises a compound as above wherein one or more of R11, R12, R13, R14 and R15 independently represents CF3, halogen, COOH, CH2COOR16, CON(R16)2, NHSO2R16, CO2R16, CO2N(R16)2, SO2N(R16)2, CONHSO2R16; SO2OR16 or aryl optionally substituted with NHCOR7, CO2R7, xe2x80x94CON(R7)2, xe2x80x94NHSO2xe2x80x94R7, xe2x80x94SO2N(R7)2 wherein R7 is as defined above; or R11 and R12 together with the phenyl group to which they are attached forms a 6-membered unsaturated ring; R16 and R7 is as defined above;
Y preferentially represents xe2x80x94NHCONHxe2x80x94;
In a preferred aspect of the invention
Y represents xe2x80x94NHCONHxe2x80x94; R1 represents tetrazolyl, and R3 represents halogen, in particular Bromo; or R3 represents phenyl which is substituted by SO2N(R16)2 wherein R16 is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl or R16 comprises a 5-8 membered ring optionally containing double bonds and optionally containing one heteroatom, or (R16)2 together with the heteroatom to which it is connected represents a 5-8 membered ring optionally containing double bonds and optionally containing another heteroatom which can be substituted by alkyl or acyl;
In yet another preferred aspect of the invention Y represents xe2x80x94NHCONHxe2x80x94; R12 represents CF3, or halogen, in particular bromo or chloro; R13 represents hydrogen or halogen in particular chloro; and R14 represents hydrogen or CF3;
The most preferred aspect of the invention comprises the following compounds:
N-phenyl-Nxe2x80x2-(2-carboxyphenyl) urea;
N-(3-trifluoromethylphenyl)-Nxe2x80x2-(2-carboxyphenyl)-N-methyl urea;
N-(3-trifluoromethylphenyl)-Nxe2x80x2-(4-bromo-2-carboxyphenyl)urea;
N-(3-trifluoromethylphenyl)-Nxe2x80x2-(2-carboxy-4-chlorophenyl)urea;
N-(3-trifluoromethylphenyl)-Nxe2x80x2-(2-carboxy-4-fluorophenyl)urea;
N-(3-bromophenyl)-Nxe2x80x2-(2-(1-H-tetrazol-5-yl)-4-biphenyl)urea;
N-(3-trifluoromethylphenyl)-Nxe2x80x2-(4xe2x80x2-(N,N-dimethylsulfamoyl)-2-(1-H-tetrazol-5-yl)-4-biphenyl)urea;
N-(3-bromophenyl)-Nxe2x80x2-(4xe2x80x2-(N,N-dimethylsulfamoyl)-2-(1-H-tetrazol-5-yl)-4-biphenyl)urea;
N-(3-bromophenyl)-Nxe2x80x2-(4xe2x80x2-(N,N-dimethylcarbamoyl)-2-(1-H-tetrazol-5-yl)-4-biphenyl)urea;
N-(3-trifluromethylphenyl)-Nxe2x80x2-(4-amino-2-(1-H-tetrazol-5-yl)phenyl)urea;
N-(3-trifluoromethylphenyl)-Nxe2x80x2-(4-acetylamino-2-(1-H-tetrazol-5-yl)phenyl)urea;
N-(3-trifluoromethylphenyl)-Nxe2x80x2-(4xe2x80x2-carbamoyl-2-(1-H-tetrazol-5-yl)-4-biphenyl)urea;
N-(3-trifluoromethylphenyl)-Nxe2x80x2-(4xe2x80x2-(N,N-dimethylcarbamoyl)-2-(1-H-tetrazol-5-yl)-4-biphenyl)urea;
N-(3-trifluoromethylphenyl)-Nxe2x80x2-(4xe2x80x2-carboxy-2-(1-H-tetrazol-5-yl)-4-biphenyl)urea;
N-(3-trifluoromethylphenyl)-Nxe2x80x2-(4xe2x80x2-(N-phenylcarbamoyl)-2-(5-tetrazoyl)-4-biphenyl)urea;
N-(2-indan)-Nxe2x80x2-(2-(1-H-tetrazol-5-yl)phenyl)urea;
N-(4-biphenyl)-Nxe2x80x2-(2-(1-H-tetrazol-5-yl)phenyl)urea;
N-(3-biphenyl)-Nxe2x80x2-(2-(1-H-tetrazol-5-yl)phenyl)urea;
N-(3-acetylphenyl)-Nxe2x80x2-(2-(1-H-tetrazol-5-yl)phenyl)urea;
N-(3-trifluoromethylphenyl)-Nxe2x80x2-(2-(1-methyltetrazol-5-yl)-4-biphenyl)urea;
N-(3-biphenyl)-Nxe2x80x2-(4-bromo-2-(1-H-tetrazol-5-yl)phenyl)urea;
N-(3-(3-pyridyl)phenyl)-Nxe2x80x2-(4-bromo-2-(1-H-tetrazol-5-yl)phenyl)urea hydrochloride;
N-(3-bromophenyl)-Nxe2x80x2-(4-bromo-2-(1-H-tetrazol-5-yl)phenyl)urea;
N-(3-bromophenyl)-Nxe2x80x2-[3xe2x80x2-nitro-2-(1-H-tetrazol-5-yl)-biphenyl]urea;
N-(3-bromophenyl)-Nxe2x80x2-[4xe2x80x2-(sulfoamido-N-methylpiperazinium chloride)-2-(1-H-tetrazol-5-yl)4-biphenyl]urea;
N-(3-bromophenyl)-Nxe2x80x2-[4xe2x80x2-carbamoyl-N-methylpiperazine)-2-(1-H-tetrazol-5-yl)-4xe2x80x2-biphenyl]urea;
N-(3,5-dichlorophenyl)-Nxe2x80x2-[4-bromo-2-(1H-tetrazol-5-yl)phenyl]urea;
N-(4-trifluoromethylphenyl-Nxe2x80x2-[4-bromo-2-(1-H-tetrazol-5-yl)phenyl]urea;
N-(4-bromophenyl)-Nxe2x80x2-[4-bromo-2-(1-H-tetrazol-5-yl)phenyl]urea;
N-(3-methoxyphenyl)-Nxe2x80x2-[4-bromo-2-(1-H-tetrazol-5-yl)phenyl]urea;
N-(3-chlorophenyl)-Nxe2x80x2-[4-bromo-2-(1-H-tetrazol-5-yl)phenyl]urea;
N-(3-methylphenyl)-Nxe2x80x2-[4-bromo-2-(1-H-tetrazol-5-yl)phenyl urea;
N-(3,4-dichlorophenyl)-Nxe2x80x2-[4-bromo-2-(1-H-tetrazol-5-yl)phenyl]urea;
N-(2-naphthyl)-Nxe2x80x2-[4-bromo-2-(1-H-tetrazol-5-yl)phenyl]urea;
N-(4-methyl-3-nitrophenyl)-Nxe2x80x2-[4-bromo-2-(1-H-tetrazol-5-yl)phenyl]urea;
N-(2-chloro-4-trifluoromethylphenyl)-Nxe2x80x2-[4-bromo-2-(1-H-tetrazol-5-yl)phenyl]urea;
N-(3,5-di(trifluoromethyl)phenyl)-Nxe2x80x2-[4-bromo-2-(1-H-tetrazol-5-yl)phenyl]urea;
N-(3,5-dimethylphenyl)-Nxe2x80x2-[4-bromo-2-(1-H-tetrazol-5-yl)phenyl]urea;
N-(4-ethoxyophenyl)-Nxe2x80x2-[4-bromo-2-(1-H-tetrazol-5-yl)phenyl]urea;
N-(4-methoxyphenyl)-Nxe2x80x2-[4-bromo-2-(1H-tetrazol-5-yl)phenyl]urea;
N-(2-trifluoromethylphenyl)-Nxe2x80x2-[4-bromo-2-(1-H-tetrazol-5-yl)phenyl]urea;
N-(2-bromophenyl)-Nxe2x80x2-[4-bromo-2-(1-H-tetrazol-5-yl)phenyl]urea;
N-(2-chlorophenyl)-Nxe2x80x2-[4-bromo-2-(1-H-tetrazol-5-yl)phenyl]urea;
N-(2-fluorophenyl)-Nxe2x80x2-[4-bromo-2-(1-H-tetrazol-5-yl)phenyl]urea;
N-(4-chloro-3-trifluoromethylphenyl)-Nxe2x80x2-[4-bromo-2-(1H-tetrazol-5-yl)phenyl]urea;
N-(3-bromophenyl)-Nxe2x80x2-(2,3-difluorophenyl)urea;
N-(2-methylphenyl)-Nxe2x80x2-[4-bromo-2-(1-H-tetrazol-5-yl)phenyl]urea;
N-(2-ethylphenyl)-Nxe2x80x2-[4-bromo-2-(1-H-tetrazol-5-yl)phenyl]urea;
N-(4-methylphenyl)-Nxe2x80x2-[4-bromo-2-(1-H-tetrazol-5-yl)phenyl]urea;
N-(2-nitrophenyl)-Nxe2x80x2-[4-bromo-2-(1-H-tetrazol-5-yl)phenyl]urea;
N-(3-fluorophenyl)-Nxe2x80x2-[4-bromo-2-(1-H-tetrazol-yl)phenyl]urea;
N-(4-[2-propyl]phenyl)-Nxe2x80x2-[4-bromo-2-(1-H-tetrazol-5-yl)phenyl]urea;
N-(3-nitrophenyl)-Nxe2x80x2-[4-bromo-2-(5-tetrazol-5-yl)phenyl]urea;
N-(3-acetylphenyl)-Nxe2x80x2-[4-bromo-2-(1-H-tetrazol-5-yl)phenyl]urea;
N-(4-nitrophenyl)-Nxe2x80x2-[4-bromo-2-(1-H-tetrazol-5-yl)phenyl]urea;
and pharmaceutically acceptable addition salts of any of the compounds above;
Examples of pharmaceutically acceptable addition salts of the compounds of the invention include inorganic and organic acid addition salts such as the hydrochloride, hydrobromide, phosphate, nitrate, perchlorate, sulfate, citrate, lactate, tartrate, maleate, fumarate, mandelate, benzoate, ascorbate, cinnamate, benzenesulfonate, methanesulfonate, stearate, succinate, glutamate, glycollate, toluene-p-sulphonate, formate, malonate, naphthalene-2-sulphonate, salicylate and the acetate. Such salts are formed by procedures well known in the art.
Other acids such as oxalic acid, while not in themselves pharmaceutically acceptable, may be useful in the preparation of salts useful as intermediates in obtaining compounds of the invention and their pharmaceutically acceptable acid addition salts.
Halogen is fluorine, chlorine, bromine or iodine.
Alkyl means a straight chain or branched chain of one to six carbon atoms, including but not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, and hexyl; methyl, ethyl, propyl and isopropyl are preferred groups.
Alkoxy is O-alkyl, wherein alkyl is as defined above.
Amino is NH2 or NH-alkyl or N-(alkyl)2, wherein alkyl is as defined above.
Acyl is xe2x80x94CO-alkyl wherein alkyl is as defined above.
Aryl is an aromatic hydrocarbon such as phenyl and naphtyl;
The acidic functional group having a pKa below 8 or a group which is converted in vivo to such group are groups such as 3-hydroxy-4-oxo-pyranyl, 2-hydroxy-4-oxo-pyrimidyl, 3,5-dioxo-1,2,4-oxadiazolidinyl, 2,4-dioxo-imidazolidinyl, 2,5-dioxo-3-hydroxy-pyrrolyl, 2,5-dioxo-pyrrolidinyl, 2,4-dioxo-1,3-thiazolidinyl, 3-hydroxy-isoxazolyl, 5-hydroxy-isoxazolyl, 3-hydroxy-isothiazolyl, 3-hydroxy-1,2,5-thiadiazolyl, tetrazolyl, 3-hydroxy-triazolyl, 3-hydroxy-pyrazolyl, 2-hydroxy-1,3,4-oxadiazolyl and 2-hydroxy-3,4-dioxo-cyclobutenyl, NH2, xe2x80x94COOR16, xe2x80x94CH2COOR16, xe2x80x94CON(R16)2, xe2x80x94NHSO2xe2x80x94R16, xe2x80x94SO2N(R16)2, xe2x80x94SO2OR16, xe2x80x94PO3H2, xe2x80x94PO3RH, xe2x80x94PO2NH2, xe2x80x94CONHOH, xe2x80x94CONHCN, xe2x80x94CONHSO2R16 and xe2x80x94CONHNH2, wherein R16 is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, alkenyl, alkynyl, aryl or aralkyl, heteroaryl or R16 comprises a 5-8 membered ring optionally containing double bonds and optionally containing one or two heteroatoms, or R16 together with the heteroatom to which it is connected form a 5-8 membered ring optionally containing double bonds and optionally containing another heteroatom;
Heteroaryl is a 5- or 6-membered heterocyclic monocyclic group. Such a monocyclic heteroaryl group includes, for example, oxazol-2-yl, oxazol-4-yl, oxazol-5-yl, isoxazol-3-yl, isoxazol-4-yl, isoxazol-5-yl, thiazol-2-yl, thiazol-4-yl, thiazol-5-yl, isothiazol-3-yl, isothiazol4-yl, isothiazol-5-yl, 1,2,4-oxadiazol-3-yl, 1,2,4-oxadiazol-5-yl, 1,2,4-thiadiazol-3-yl, 1,2,4-thiadiazol-5-yl, 1,2,5-oxadiazol-3-yl, 1,2,5-oxadiazol-4-yl, 1,2,5-thiadiazol-3-yl, 1,2,5-thiadiazol-4-yl, 1-imidazolyl, 2-imidazolyl, 4-imidazolyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 2-furanyl, 3-furanyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 3-pyridazinyl, 4-pyridazinyl, 2-pyrazinyl, 1-pyrazolyl, 3-pyrazolyl, and 4-pyrazolyl.
A 5-8 membered ring optionally containing double bonds and optionally containing one or two heteroatoms includes for example pyrrolidine, piperidine, piperazine, morpholine, cyclohexyl, cyclohexen, dihydropyrrole, dihydrofuran, dihydrothiophen, dihydropyridine, dihydropyridazine, dihydropyrimidine, dihydropyrazine, tetrahydropyridine, tetrahydropyridazine, tetrahydropyrimidine, tetrahydropyrazine, homopiperazine, homopiperidine, azacyclooctane.
The compounds of this invention may exist in unsolvated as well as in solvated forms with pharmaceutically acceptable solvents such as water, ethanol and the like. In general, the solvated forms are considered equivalent to the unsolvated forms for the purposes of this invention.
It will be appreciated by those skilled in the art that the compounds of the present invention contain several chiral centres and that such compounds exist in the form of isomers (i.e. enantiomers). The invention includes all such isomers and any mixtures thereof including racemic mixtures.
Some of the compounds of the present invention exist in (+) and (xe2x88x92) forms as well as in racemic forms. Racemic forms can be resolved into the optical antipodes by known methods, for example, by separation of diastereomeric salts thereof with an optically active acid, and liberating the optically active amine compound by treatment with a base. Another method for resolving racemates into the optical antipodes is based upon chromatography on an optically active matrix. Racemic compounds of the present invention can thus be resolved into their optical antipodes, e.g., by fractional crystallization of d- or l-(tartrates, mandelates, or camphorsulphonate) salts for example. The compounds of the present invention may also be resolved by the formation of diastereomeric amides by reaction of the compounds of the present invention with an optically active activated carboxylic acid such as that derived from (+) or (xe2x88x92) phenylalanine, (+) or (xe2x88x92) phenylglycine, (+) or (xe2x88x92) camphanic acid or by the formation of diastereomeric carbamates by reaction of the compounds of the present invention with an optically active chloroformate or the like.
Additional methods for the resolvation of optical isomers, known to those skilled in the art may be used, and will be apparent to the average worker skilled in the art. Such methods include those discussed by J. Jaques, A. Collet, and S. Wilen in xe2x80x9cEnantiomers, Racemates, and Resolutionsxe2x80x9d, John Wiley and Sons, New York (1981).
The compounds of the invention may be prepared in numerous ways. The compounds of the invention and their pharmaceutically acceptable derivatives may thus be prepared by any method as discribed above or known in the art for the preparation of compounds of analogous structure, and as shown in the representative examples which follow.
The compounds of the present invention are potent blockers of chloride channels in normal as well as sickle cell erythrocytes. The ability of the compounds to block the erythrocyte chloride channels can not be demonstrated by classical electrophysiological measurements such as patch clamping, since the channel unit conductance is below the detection limit of these techniques.
All dose-response experiments were therefore performed by concomitant measurements of conductive netfluxes of Cl31  (JCl) and membrane potentials (Vm) in suspensions of erythrocytes (Bennekou, P. and Christophersen, P. (1986), Flux ratio of Valinomycinxe2x80x94Mediated K+ Fluxes across the Human Red Cell Membrane in the presence of the Protronophore CCCP. J. Membrane Biol. 93, 221-227.). The membrane Cl31  conductances (GCl) were calculated by the following equation (Hodgkin, A. L. and Huxley, A. F. (1952) The components of membrane conductance in the giant axon of Loligo. J. Physiol. Lond. 116, 449-472):       G    Cl    =            F      *              J        Cl                    (                        V          m                -                  E          Cl                    )      
where F is the Faraday constant, ECl is the Nernst potential for the Cl-ion. Administration of 3-Trifluoromethylphenyl-2-carboxyphenyl urea to a suspension of normal erythrocytes blocked GCl more than 95% with a KD-value of 1.3 xcexcM. The compound equipotently blocked GCl from oxygenated as well as deoxygenated homozygoteous sickle cell erythrocytes.
The KD-value for for N-(3-Trifluoromethylphenyl)-Nxe2x80x2-(4xe2x80x2-(N,N-dimethylsulfamoyl)-2-(1-H-tetrazol-5-yl)-4-biphenyl)urea in this test was 0.3 xcexcM.
The compounds of the present invention are useful as blockers of the bone degrading activity of osteoclasts. For measuring the activity of the compounds different osteoclast inhibition assays known in the art can be used.
While it is possible that, for use in therapy, a compound of the invention may be administered as the raw chemical, it is preferable to present the active ingredient as a pharmaceutical formulation. Optionally the compounds of the invention can be administered together with another therapeutic compound. This can optionally be in the form of a pharmaceutical composition containing more than one compound.
The invention thus further provides pharmaceutical formulations comprising a compound of the invention or a pharmaceutically acceptable salt or derivative thereof together with one or more pharmaceutically acceptable carriers therefor and, optionally, other therapeutic and/or prophylactic ingredients. The carrier(s) must be xe2x80x9cacceptablexe2x80x9d in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
Pharmaceutical formulations include those suitable for oral, rectal, nasal, topical (including buccal and sub-lingual), vaginal or parenteral (including intramuscular, sub-cutaneous and intravenous) administration or in a form suitable for administration by inhalation or insufflation.
The compounds of the invention, together with a conventional adjuvant, carrier, or diluent, may thus be placed into the form of pharmaceutical compositions and unit dosages thereof, and in such form may be employed as solids, such as tablets or filled capsules, or liquids such as solutions, suspensions, emulsions, elixirs, or capsules filled with the same, all for oral use, in the form of suppositories for rectal administration; or in the form of sterile injectable solutions for parenteral (including subcutaneous) use. Such pharmaceutical compositions and unit dosage forms thereof may comprise conventional ingredients in conventional proportions, with or without additional active compounds or principles, and such unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be employed. Formulations containing ten (10) milligrams of active ingredient or, more broadly, 0.1 to one hundred (100) milligrams, per tablet, are accordingly suitable representative unit dosage forms.
The compounds of the present invention can be administrated in a wide variety of oral and parenteral dosage forms. It will be obvious to those skilled in the art that the following dosage forms may comprise, as the active component, either a compound of the invention or a pharmaceutically acceptable salt of a compound of the invention.
For preparing pharmaceutical compositions from the compounds of the present invention, pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances which may also act as diluents, flavouring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.
In powders, the carrier is a finely divided solid which is in a mixture with the finely divided active component.
In tablets, the active component is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired.
The powders and tablets preferably contain from five or ten to about seventy percent of the active compound. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. The term xe2x80x9cpreparationxe2x80x9d is intended to include the formulation of the active compound with encapsulating material as carrier providing a capsule in which the active component, with or without carriers, is surrounded by a carrier, which is thus in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid forms suitable for oral administration.
For preparing suppositories, a low melting wax, such as admixture of fatty acid glycerides or cocoa butter, is first melted and the active component is dispersed homogeneously therein, as by stirring. The molten homogenous mixture is then poured into convenient sized molds, allowed to cool, and thereby to solidify.
Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or sprays containing in addition to the active ingredient such carriers as are known in the art to be appropriate.
Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water-propylene glycol solutions. For example, parenteral injection liquid preparations can be formulated as solutions in aqueous polyethylene glycol solution.
The compounds according to the present invention may thus be formulated for parenteral administration (e.g. by injection, for example bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilising and/or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilisation from solution, for constitution with a suitable vehicle, e.g. sterile, pyrogen-free water, before use.
Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavours, stabilizing and thickening agents, as desired.
Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, or other well known suspending agents.
Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for oral administration. Such liquid forms include solutions, suspensions, and emulsions. These preparations may contain, in addition to the active component, colorants, flavours, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.
For topical administration to the epidermis the compounds according to the invention may be formulated as ointments, creams or lotions, or as a transdermal patch. Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilising agents, dispersing agents, suspending agents, thickening agents, or colouring agents.
Formulations suitable for topical administration in the mouth include lozenges comprising active agent in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
Solutions or suspensions are applied directly to the nasal cavity by conventional means, for example with a dropper, pipette or spray. The formulations may be provided in single or multidose form. In the latter case of a dropper or pipette, this may be achieved by the patient administering an appropriate, predetermined volume of the solution or suspension. In the case of a spray, this may be achieved for example by means of a metering atomising spray pump.
Administration to the respiratory tract may also be achieved by means of an aerosol formulation in which the active ingredient is provided in a pressurised pack with a suitable propellant such as a chlorofluorocarbon (CFC) for example dichlorodifluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. The aerosol may conveniently also contain a surfactant such as lecithin. The dose of drug may be controlled by provision of a metered valve.
Alternatively the active ingredients may be provided in the form of a dry powder, for example a powder mix of the compound in a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidone (PVP). Conveniently the powder carrier will form a gel in the nasal cavity. The powder composition may be presented in unit dose form for example in capsules or cartridges of, e.g., gelatin, or blister packs from which the powder may be administered by means of an inhaler.
In formulations intended for administration to the respiratory tract, including intranasal formulations, the compound will generally have a small particle size for example of the order of 5 microns or less. Such a particle size may be obtained by means known in the art, for example by micronization. When desired, formulations adapted to give sustained release of the active ingredient may be employed.
The pharmaceutical preparations are preferably in unit dosage forms. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packaged tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.
Tablets or capsules for oral administration and liquids for intravenous administration are preferred compositions.
The compounds of the present invention are very useful in the treatment of sickle cell anaemia, brain oedema following ischaemia or tumors, diarrhea, hypertension (diuretic), bone metabolic disorders, osteoclast associated disorders and glaucoma, due to their potent chloride channel blocking activity. These properties make the compounds of this invention extremely useful in the treatment of sickle cell anaemia, brain oedema following ischaemia or tumors, diarrhea, hypertension (diuretic), bone metabolic disorders, osteoclast associated disorders and glaucoma, as well as other disorders sensitive to the chloride channel blocking activity of the compounds of the present invention. The compounds of this invention may accordingly be administered to a living animal body, including a human, in need of treatment, alleviation, or elimination of an indication associated with or responsive to chloride channel blocking activity. This includes especially sickle cell anaemia, brain oedema following ischaemia, or tumors, diarrhea, hypertension (diuretic), bone metabolic disorders, osteoclast associated disorders and glaucoma.
Suitable dosage range are 0.1-500 milligrams daily, and especially 10-70 milligrams daily, administered once or twice a day, dependent as usual upon the exact mode of administration, form in which administered, the indication toward which the administration is directed, the subject involved and the body weight of the subject involved, and further the preference and experience of the physician or veterinarian in charge. When administered in combination with compounds known in the art for treatment of the diseases, the dosis regimen may be reduced.
The invention then comprises alone or in combination, the following:
the use of any of the compounds as mentioned above for the preparation of a medicament for the treatment of sickle-cell anaemia, brain oedema following ischaemia, or tumours, diarrhea, hypertension (diuretic), bone metabolism disorders, glaucoma, allergic or inflammatory conditions or healing ulcers;
the use of any of the compounds as above combined with the use of other bone metabolism controlling compounds for the treatment of bone metabolic disorders or conditions.
the use as above wherein the known compounds are bisphophonates such as etidronate, pamidronate, or clodronate optionally combined with calcium; oestrogen-receptor active compounds such as oestrogen i.e. oestradiol and ethyloestradiol, calcitonin, 1,25-dihydroxyvitamine D and metabolites thereof, fluoride, growth hormone, parathyreoidea hormone, triiodo-thyrosine, protease inhibitors ie. collagen degrading enzymes, or cancer therapeutic agents.
a method for the treatment of a disorder or disease of a living animal body which disorder or disease is sickle-cell anaemia, brain oedema following ischaemia or tumours, diarrhea, hypertension (diuretic), bone metabolic disorders, glaucoma, allergic or inflammatory conditions or ulcers, comprising administering to such a living animal body, including a human, in need thereof a therapeutically effective amount of a compound as any above;
a method for the treatment of a disorder or disease of a living animal body which disorder or disease is a bone metabolic disorder comprising administering to such a living animal body, including a human, in need thereof a therapeutically effective amount of a compound as any above optionally in combination with other bone metabolism controlling compounds.
In the context of this invention a bone metabolic disorder or disease covers any deviation in the bone formation and/or bone resorption as ie. osteoclast associated diseases or disorders, age related decrease in bone mass, osteoporosis, osteopeni, osteogenesis imperfecta, osteopetrosis, osteosklerosis, Paget""s disease of bone, bone metastasizing cancers, osteomyelitis, osteonekrosis, fluorosis, bone malignancies etc. and covers in general all clinical types of the mentioned diseases;
In the context of this invention the compounds known for treatment for bone metabolic disorders includes bisphophonates such as etidronate, pamidronate, oestrogen-receptor binding compounds such as oestrogen i.e. oestradiol and ethyloestradiol; calcitonin, 1,25-dihydroxyvitamine D and metabolites thereof, fluoride, growth hormone, parathyroid hormone, triiodo-thyrosine, cancer therapeutic agents and inhibitors of collagen degrading enzymes such as protease inhibitors.
In the context of this invention the method of treatment of a disorder or disease of a living animal body including a human comprising administering a compound of the invention optionally together with a known compound and optionally administerering the compound(s) in a reduced dosis;
The treatment of the diseases and disorder can be in chronical or a long term treatment as well as a treatment of sudden crisis in the disease and disorder.
The following examples will illustrate the invention further, however, they are not to be construed as limiting.